Browsing by Subject "long-read genomics"

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    Microfluidic Dna Sample Preparation For Long-Read Genomics
    (2020-05) Agrawal, Paridhi
    With the commercialization of genomics technologies, DNA sequencing has become an affordable and accessible tool for innumerable biological advancements. The fast speed, low cost, automated measurement and high throughput nature of these sophistically engineered miniaturized systems is made possible by the rapid advancement in microfluidics. Owing to superior fabrication capabilities and adequate handling of complex samples, microfluidic systems have shown promise for varied biological applications. While measurements are performed at the micro scale in all genomics systems, DNA extraction and pre-processing are done externally, resulting in a wide mismatch between the amount of sample prepared and the amount utilized. This work focuses on using microfluidics as a tool to assist, and hopefully improve, genomics methods. Long-read genomics technologies are capable of obtaining long-range information from DNA molecules about repetitive and complex regions of the genome. Optimal application of these technologies requires shear-free methods for extracting long DNA from cells. These sample preparation tools should be facile, inexpensive, universal and amenable to automation. In addition to providing all these capabilities, microfluidics can not only expedite sample preparation, but also offer the opportunity for direct upstream integration to eliminate DNA fragmentation and loss during transfer to the genomic device. The work outlined here presents a microfluidic platform for long DNA sample preparation. In the 3D cell culture-inspired proof-of-principle poly(dimethylsiloxane) device, gel-based high molecular weight DNA extraction and continuous flow purification is followed by electrophoretic extraction of the long DNA from the miniaturized gel. The device successfully demonstrated extraction of DNA as long as 4 megabase pairs from cells, but the 10 ng DNA yield was insufficient for some genomics experiments. A scaling up of the device design, realized by 3D printing, resulted in a high-yield next-generation device which completely eliminates cleanroom fabrication, making the method accessible to users outside the microfluidics community. The 100 ng DNA extracted from the next-generation device were used for size analysis in commercial genome mapping nanochannels. Along with competitive yield and DNA sizes, the miniaturized format reduces the standard day-long DNA extraction process to a few hours, making it a promising prototype platform for routine long DNA sample preparation. The generic device design and straightforward protocol provide integration and automation capabilities to the platform presented, which are absent in existing alternatives to the plug lysis method. Future avenues of development and application are hypothesized to fully realize the potential of the sample preparation platform. The continued engineering and genomics upgrades justify the proposed strategies.